<B>Regulation of gene transcription</B> Genetic regulatory mechanisms (i). <i>Transcription initiation</i>. The ongoing projects described during the last site visit on steps of open complex formation, promoter melting and the role of -11A (master base) in isomerization as assayed by 2, AP fluorescence were completed and published. We are currently working on identifying the amino acid residue(s) in the RNA polymerase that the master base (-llA) contacts to signal further base-pair opening. (ii). <i>Mechanism of repression of galP1by GalR</i> We previously hypothesized that GalR represses the <i>galP1</i> promoter by making a direct protein-protein contact with a promoter-bound RNA polymerase to stabilize the RNA polymerase-DNA complex thereby increasing the activation energy of the isomerization step. As proposed, we were able to isolate a GalR mutant which binds to DNA but fails to contact RNA polymerase (negative control, <i>nc</i>, mutant). This work, supporting the contact-inhibition model of transcription repression was published. We are currently working on cross-linking GalR to RNA polymerase using the <i>nc</i> mutant as a control in order to identify interfaces. (iii). <i>A new promoter element in lac</i>. We isolated a hyperactive mutant of the <i>lac</i> promoter, called <i>P1-6</i>, defined a new element at the 15 region of the promoter, and determined the kinetic behavior of RNA polymerase at this promoter. These results have been published. Currently, we are determining the contact point of the 15 DNA sequence of the promoter to RNA polymerase. (iv). <i>Promoter clearance</i>. Previously we proposed that LacI repressor, which represses transcription initiation at <i>P1-6</i> by blocking RNA polymerase binding, represses transcription at a <i>lacP1-6</i> variant by blocking promoter clearance. Evidence supporting the model have been published. We are currently studying the kinetics of this process. (v). <i>Rules of transcription start point selection</i>. We defined the axiom of determining the transcription start point (tsp) in the two <i>gal</i> promoters. We published these results, and then extended the investigation to the tsp selection in the hyperactive <i>P1-6</i> mentioned above. A manuscript describing the latter will be submitted shortly. (vi). <i>Effect of supercoiling on promoter activities</i>. Our studies on the influence of the degree of negative supercoiling on different promoters have been published. (vii). <i>Repressosome structure</i>. We continued to study the structure of GalR repressosome complex, which contains a DNA loop and the architectural protein HU. We proposed four possible DNA trajectories in a repressosome. From energetic calculations, atomic force microscopy, and molecular genetic experiments, we concluded that the GalR repressosome has an antiparallel (AL1, one of the four) DNA trajectories. The recent results have been published in two papers. Interestingly, an antiparallel orientation has since been shown in other DNA loop systems that have been studied. We are now studying the DNA trajectory using single DNA molecule system. We also published biochemical and the biophysical characterizations of the different sub-components of the <i>gal</i> repressosome. (viii). <i>Single DNA molecule studies of the gal-DNA loop</i>. Most of our work on determining kinetic and thermodynamic parameters of <i>gal</i> DNA loop using single DNA molecules (collaboration with Dr. Laura Finzi) have been published. Other single molecule investigations are described in the Research Summary. (ix). <i>X-ray structure of HU</i>. We determined the structure of <i>E. coli</i> HUalphabeta heterodimer by xray diffraction. Interestingly, it shows a stacked spiral structure with an octameric repeating unit, and with the DNA binding beta-loops of the protein projected outward convenient for a DNA wrap-around. Since the HU spiral could be left- or right handed, the wrap-around DNA could give rise to both negative and positive supercoiling. We published the structure, and currently studying the role of the HU spiral structure in explaining the HUs role in creating and retaining DNA superhelicity. (x). <i>Role of HU in determining global transcription pattern</i>. We isolated and characterized an HUalpha mutant of <i>E. coli</i> which changes the gene expression pattern and cell morphology in a global fashion. Our results suggest that the mutant HU does so by altering the DNA structure in an unknown way. This work has been published. We are currently studying the nature of changes that the mutant HU induces in DNA and how that alters the transcription profile of <i>E. coli</i> promoters. (xi). <i>Bacterial nucleoid</i>. Bacterial DNA is compacted about 1000-fold in the nucleoid. Our ability to distinguish between antiparallel (loop) and parallel (solenoid) DNA trajectories, and our finding that the nucleoid HU protein, can change global gene expression by changing DNA structure, prompted us to study structure-function relationships of the <i>E. coli</i> nucleoid using several different approaches. Our ongoing research on this subject are elaborated in the Research Summary section. (xii). <i>Lysogencity of bacteriophage lambda</i>. We continued to study (a) the developmental process by which phage lambda decides, after it infects the <i>E. coli</i> host, whether to follow the lytic or lysogenic pathway, and (b) the cause of extreme stability of the lambda prophage in a lysogen. Our views about lambdas decision making process (a collaboration with Dr. Donald Court and late Dr. Amos Oppenheim) have been published in a review. We are investigating the role of the phage proteins in the decision making process both by molecular genetic and DNA array experiments. One of the aims is to establish whether the decision to go lysogenic or lytic after infection of the host is stochastic (proposed by others) or deterministic (our favorite model). Our efforts are described in Research Summary. We have also proposed that the extreme stability of the lambda prophage may be because of a DNA loop formation involving the six operators (binding sites of the prophage CI repressor). We are studying DNA looping in lambda (a) by determining the kinetic and thermodynamic parameters in single DNA molecules by following tethered particle motions (collaboration with Laura Finzi), (b) by <i>in vitro</i> transcription under conditions of DNA looping, and (c) by measuring spontaneous phage production in various loop-unstable operator mutants